Specific proteolytic enzymes (proteases) play critical roles in a number of important human and animal diseases. In some cases, proteases are essential for the replication of microbial pathogens. In other cases they may have harmful effects on target tissues and organs independent of any microbial involvement.
Among the microbes that depend upon the action of specific proteases are the picornaviruses, which are currently divided into four genera: Enterovirus, Cardiovirus, Rhinovirus and Aphthovirus (foot-and-mouth disease viruses). Coxsackieviruses are picornaviruses within the Enterovirus group which cause a wide spectrum of human diseases, ranging from mild respiratory illness to more serious disseminated diseases of the nervous system and heart (Melnick, J. L., in Virology, 1985, B. N. Fields, Ed., Raven Press, New York, pp. 739-794). Although most of the detailed information on this group of viruses comes from in-depth analyses of poliovirus structure and replication, other members of the picornaviruses have the same genomic organization and appear to function similarly (Rueckert, R. R., in Virology, 1985, B. N. Fields, Ed., Raven Press, New York, pp. 705-738).
The genomes of all picornaviruses comprise a messenger-active single-stranded RNA molecule. This RNA encodes a single large polyprotein that is cleaved to produce both structural and non-structural mature viral proteins. In poliovirus, these cleavages are carried out by two virally-encoded proteases designated 2A and 3C. The 2A protease is responsible for the primary cleavage that generates the viral capsid precursor P1 [Toyoda et al., Cell 45:761 (1986)]. The 3C protease, which is itself released from the polyprotein by an autocatalytic cleavage, then carries out all but one of the remaining cleavage reactions [Hanecak et al., Cell 37:1063 (1984)]. This protease specifically cleaves the peptide bond between certain adjacent glutamine and glycine residues.
Another microbial pathogen dependent upon a specific protease is the human immunodeficiency virus type 1 (HIV-1), the causative agent of most cases of AIDS. The HIV-1 protease, which is part of the pol gene product and is instrumental in the cleavage of large precursor proteins into structural and functional viral proteins, is specific for HIV proteins [Ratner et al., Nature 313:277 (1985); Farmerie et al., Science 236:305 (1987)].
In some cases, proteases are involved in the destructive effects of microbial pathogens in ways that do not involve replicative processes. For example, Clostridium histolyticum, a bacterium that causes gas gangrene, secretes a battery of collagenases that produce multiple cleavage of collagen. These collagenases, which specifically cleave at the amino side of glycine residues in peptide linkages, contribute to the invasiveness of the bacterium by destroying the connective-tissue barriers of the host. The bacterium itself is unaffected by its secretory products because it, unlike the multicellular eukaryotes it infects, is devoid of collagen.
Destructive effects of collagenases have also been implicated in disease states which do not involve microbial pathogens. For example, the destruction of connective tissue collagen in bone joints by collagenases in some forms of arthritis is known to occur.
Because the above-mentioned proteases and others are involved in many human and animal diseases, it would be desirable to identify potential inhibitors of the specific proteases involved. Screening assays that can be used to identify such inhibitors, however, are presently laborious to carry out and/or relatively insensitive.